Some wild type viruses, such as mumps virus, measles virus, influenza virus, HIV, West Nile Virus, smallpox virus, and parainfluenza virus have a significant potential for infecting and damaging the nervous system (i.e., neurotoxicity). Live, attenuated vaccines derived from these and other related viruses must be sufficiently attenuated to reduce the risk of neurotoxic adverse events (AE). In order to identify safer vaccines, pre-clinical neurotoxicity tests need to be developed to screen experimental vaccine candidates prior to use in humans as well as to determine manufacturing consistency (i.e., when new vaccine master seed stocks are derived). Because neurotoxicity is a disease syndrome dependent upon the host's genetic and developmental state (i.e., vaccines for pediatric use), we include in our research efforts studies of the effects of viruses on the developing nervous system. Since we lack sufficient information about the pathophysiological responses to viruses that infect the nervous system, we have developed an animal model of wild type virus-induced developmental damage that serves as the first virus-associated model for autism, and have used this information to develop neurotoxicity test paradigms, including mumps, measles, influenza, and vaccinia-based smallpox vaccines. Using the basic discoveries from these and other studies, we develop research-based specific neurotoxicity tests for these and other virus vaccines initially with animal models (with behavioral, anatomical, neurochemical, and molecular biological endpoints) to evaluate risk for neurotoxicity or relative attenuation for the nervous system. Once the animal disease model is developed, our goal is to develop cell culture or molecular biological assays to test for virus vaccine neurotoxicity. Following development of the research-based neurotoxicity test, we seek international regulatory laboratory collaborations to evaluate the potential regulatory use of the test paradigms. Smallpox Vaccine Safety: Development and Validation of Pre-Clinical Toxoclogy Tests for Vaccinia-based Smallpox Vaccine through Molecular Mechanism of Vaccine Neurotoxology. Smallpox virus is a Category A bioterrorism/biowarfare agent. The licensed vaccinia virus-based vaccine (Dryvax) is effective, but produces significant and serious adverse events (AE) (Lane, 1969; Vega, 1969; Adams, 1973; Terzin, 1974; Edis, 1975; Gurvich, 1975; Kurata, 1977); one of the most deadly AEs is central nervous system (CNS) disease, with children being at greater risk than adults. Fortunately, new, and, it is hoped, safer smallpox vaccines are in development. As a standard regulatory consideration, pre-clinical neurotoxicity assays are used as in an attempt to predict the risk of damage to the human CNS (neurotoxicity) from live virus vaccines; no such validated neurotoxicity assay is available for vaccinia-based smallpox vaccines. To avoid the expense and validation problems inherent in primate testing, we have developed a prototype smallpox vaccine neurotoxicity assay using rodents. Preliminary data indicate this assay can discriminate differences in vaccinia virus strain-specific neurotoxicity among smallpox vaccines (Dryvax, Lister), and laboratory strains (WR, MVA), with WR>Lister>Dryvax>MVA in order of decreasing neuro-toxicity. Here we propose to 1) complete development and validation of the in vivo mouse neurotoxicity assay as a standardized regulatory safety test to expedite the licensing of safer smallpox vaccines, and 2) use this assay as a disease model to study the molecular pathogenesis of vaccinia-based smallpox vaccine neurotoxicity by identifying critical virus-neural cell gene/gene-product interactions. These studies will improve smallpox vaccine safety tests, e.g., small animal, in vitro and molecular biological-based neurotoxicity assays, and can promote new vaccine development, and e.g., rational attenuation of smallpox vaccines via targeted mutations. PROGRESS. We have developed and tested a neonatal mouse model for assessing the relative toxicity of vaccinia virus strains. We have also made progress towards achieving the second objective of the project by identifying the viral and host gene interactions in adult and infant using human brain tissue-derived cDNA libraries. We are currently assessing the functional role of these interactions. Vaccine Safety: Pathogenesis of virus vaccine neurotoxicity. a) Molecular Markers of Neurotoxicity: We have identified mutations in the mumps virus genome associated with increased and decreased risk of neurotoxicity. b) Animal Models of CNS Diseases: 1) Autism. Viruses are known etiologic agents of autism (e.g., intrautarine infection with rubella virus). Therefore, concerns are raised by the public regarding a possible relationship between childhood vaccines and autism. Because no valid animal models existed to study the pathogenesis of the neuroanatomical and behavioral signs of autism, we have developed a rat model of autism using neonatal infection with neurotropic viruse, Borna disease virus. We have characterized autistic-like changes in neuroanatomy, neurochemistry, neurological disease and behavior in these rats. In addition, we have identified regional and developmental changes in neurotransmitters, including serotonin and norepinephrine. We have now identified host genetic background features that affect neurotoxic outcomes.We have identified genomic changes in the G and L proteins associated with changes in neurotoxicity outcomes. 2) We have established a model to study the pathogenesis of wild type measles virus in the developing mouse brain 3) We have established a model to study the pathogenesis of wild type influenza virus in the developing rat brain. Vaccine Safety: Neurotoxicity safety test development, validation and evaluation. Human brain continues to develop during the first few years of postnatal life. Since the developing brain is uniquely sensitive to damage following virus infection, administration of neurovirulent vaccines to infants can place the child's nervous system at increased risk for vaccine related injury. There is a need to develop a test to assess the vaccine's human neurotoxicity potential, in order to develop the safest vaccines possible, and to perform appropriate risk assessment/risk management, particularly for new vaccines. 1. A newborn rat mumps virus neurotoxicity test was developed we are proceeding with international validation studies with international regulatory laboratories such as NIBSC in England, Health Canada and are in preliminary stages with health authorities in Brazil. We have also discovered virus genomic changes associated with neurotoxicity alterations, as well as molecular markers of host neuronal damage following infection. 2. Although rare, CNS events associated with wild type influenza virus can occur. In this project we are developing an assay to evaluate the relative neurotoxicity of influenza viruses (wild type and vaccine) using a newborn rat model. Our data show that we can differentiate between wild type influenza virus and vaccine strains in our neurotoxicity test. 3. We have developed a newborn mouse model for differentiating the toxicity of vaccinia virus strains. We can differentiate between vaccinia strains of greater or lesser neurotoxicity, suggesting this test will have utility as a pre-clinical toxicity test for new smallpox vaccines. 4. We have developed a mouse model for measles virus neurotoxicity testing using newborn mice. This project incorporates FY2002 projects 1Z01BK002007-07 and 1Z01BK002008-07.